Abstract
A histochemical fluorescence method is presented in which a controlled activation of the condensation reaction between formaldehyde and certain aromatic monoamines is achieved by the introduction of minute amounts of HCl gas in the formaldehyde reaction vessel. In this way, not only highly reactive catecholamines and 5-hydroxytryptamine but also less reactive compounds, such as tryptamine, 5-methoxytryptamine and 3-methoxylated phenylethylamines which give only weak fluorescence in the noncatalyzed formaldehyde reaction, can be converted into intensely fluorescent products. The acid-catalyzed formaldehyde treatment proved to be a highly sensitive technique for the demonstration of these various monoamines in freeze-dried tissues. The reaction thus proceeded without any observable dislocation of the substances from their cellular stores and the resulting fluorophores were well visible against an essentially dark background. The procedure was successfully, and equally well, applied to whole freeze-dried tissue pieces, to paraffin sections of freeze-dried specimens and to freeze-dried cryostat sections. The fluorescence properties of a large number of phenylethylamine and indolylethylamine fluorophores, formed in the acid-catalyzed formaldehyde reaction, have been investigated microspectrofluorometrically in models and in freeze-dried tissues. The fluorescence spectra of the amine fluorophores were consistent and fully reproducible, making possible the accurate characterization of cellular monoamines with this method.